scholarly journals Isolation and some structural analyses of a proteodermatan sulphate from calf skin

1983 ◽  
Vol 213 (2) ◽  
pp. 289-296 ◽  
Author(s):  
T Nakamura ◽  
E Matsunaga ◽  
H Shinkai
Keyword(s):  
Core Protein ◽  
Gradient Centrifugation ◽  
Cetylpyridinium Chloride ◽  
Periodic Acid ◽  
Molar Ratio ◽  
Gel Chromatography ◽  
Dermatan Sulphate ◽  
Periodic Acid Schiff ◽  
Cscl Density Gradient ◽  
Calf Skin

A proteodermatan sulphate was isolated from 0.15 M-NaCl and 0.45 M-NaCl extracts of newborn-calf skin. The proteoglycan was separated from collagen and hyaluronic acid by precipitation with cetylpyridinium chloride and CsCl-density-gradient centrifugation. Further purification was performed by ion-exchange, affinity and molecular-sieve chromatography. The proteoglycan bound to concanavalin A-Sepharose in 1 M-NaCl. It gave a positive reaction with periodic acid/Schiff reagent and contained 8.3% of uronic acid. The dermatan sulphate, the only glycosaminoglycan component, was composed of 74% iduronosylhexosamine units and 26% glucuronosylhexosamine units. The Mr was assessed to be 15000-20000 by gel chromatography. The core protein was found to be a sialoglycoprotein that had O-glycosidic oligosaccharides with N-acetylgalactosamine at the reducing termini. The molar ratio of oligosaccharide chains to dermatan sulphate was approx. 3:1. From these results the proposed structure of proteodermatan sulphate is: one dermatan sulphate chain (average Mr 17500), three O-glycosidic oligosaccharide chains and probably N-glycosidic oligosaccharide chain(s) bound to one core-protein molecule (Mr 55000).

scholarly journals Isolation of 35S- and 3H-labelled proteoglycans from cultures of human embryonic skin fibroblasts

1979 ◽  
Vol 183 (3) ◽  
pp. 669-681 ◽  
Author(s):  
L Cöster ◽  
I Carlstedt ◽  
A Malmström
Keyword(s):  
Density Gradient Centrifugation ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Guanidine Hydrochloride ◽  
Gel Chromatography ◽  
Dermatan Sulphate ◽  
Cscl Density Gradient ◽  
Embryonic Skin ◽  
Sepharose 4B ◽  
Phosphate Buffered Saline

35SO42– and [3H]-leucine-labelled proteoglycans were isolated from the medium of a fibroblast culture, from an EDTA extract of the monolayer, and from consecutive dithiothreitol and guanidine hydrochloride extracts of the cells. Proteoglycans of different sizes were isolated from the extracts by gel chromatography on Sepharose 4B. In the medium and the EDTA extract the largest proteoglycans contained only 35S-labelled galactosaminoglycan, whereas all other fractions contained in addition heparan [35S-labelled galactosaminoglycan, whereas all other fractions contained in addition heparin [35S]sulphate. The galactosaminoglycan-containing proteoglycans of the various extracts were separated into a larger component, containing chondroitin sulphate-like side chains, and a smaller component, containing dermatan sulphate. The larger proteoglycan of the medium showed reversible association-dissociation behaviour when chromatographed on Sepharose CL2B in phosphate-buffered saline and 4M-guanidine hydrochloride respectively. This property remained after removal of extraneous proteins by CsCl-density-gradient centrifugation in guanidine hydrochloride. The association was markedly increased by the addition of high-molecular-weight hyaluronic acid.

scholarly journals Attempted isolation of a heparin proteoglycan from bovine liver capsule

1970 ◽  
Vol 116 (1) ◽  
pp. 27-34 ◽  
Author(s):  
U. Lindahl
Keyword(s):  
Potassium Chloride ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Bovine Liver ◽  
Gel Chromatography ◽  
Dermatan Sulphate ◽  
Liver Capsule ◽  
Neutral Sugars ◽  
Degraded Material ◽  
Heparin Preparation

(1) Polysaccharides were isolated from bovine liver capsule by extraction with 2m-potassium chloride followed by precipitation from 0.8m-potassium chloride with cetylpyridinium chloride. Chondroitin sulphate was eliminated by digestion with hyaluronidase. The yield of heparin was approx. 40% of that obtained after extraction of the papain-digested tissue. (2) The macromolecular properties of the hyaluronidase-digested polysaccharide were studied by gel chromatography on Sephadex G-200 of the intact, as well as of the alkali-degraded, material. The results suggested the presence of single heparin chains in addition to a dermatan sulphate proteoglycan. (3) A purified heparin preparation was analysed for amino acids and neutral sugars. Xylose, galactose and serine were found in amounts corresponding to 0.1, 0.2, and 0.4 residue/polysaccharide chain (mol.wt. 7400), respectively. It is suggested that the isolated material had been degraded by a polysaccharidase with endo-enzyme properties.

scholarly journals DISTRIBUTION OF MUCOSUBSTANCES IN THE DEVELOPING RAT HEART

1972 ◽  
Vol 20 (11) ◽  
pp. 896-907 ◽  
Author(s):  
ROGER R. MARKWALD ◽  
WILLIAM N. ADAMS SMITH
Keyword(s):  
Electrolyte Concentration ◽  
Cetylpyridinium Chloride ◽  
Periodic Acid ◽  
Frozen Sections ◽  
Concentrated Solutions ◽  
Periodic Acid Schiff ◽  
Rat Hearts ◽  
Aldehyde Fuchsin ◽  
Developing Rat ◽  
Fibroblastic Cells

Mucosubstances (MS) were examined in 10½-14½-day embryonic rat hearts utilizing nonaqueous fixatives or formaldehyde vapor-fixed frozen sections hydrated in concentrated solutions of cetylpyridinium chloride. Ribonuclease-resistant, polyanionic sites were limited to the extracellular cardiac jelly, endocardium and fibroblastic cells (cushion tissue) associated with the endocardium. The cardiac jelly and endocardium of day 10½ embryos principally contained a hyaluronic acid-like carboxylated mucosubstance whose alcianophilia at pH 2.5 was abolished by hyaluronidase but was resistant to NaOH extraction and neuraminidase and trypsin digestion. A critical electrolyte concentration of 0.2 M MgCl2 abolished alcianophilia. On days 13½-14½ carboxylated MS were restricted to cushion tissue and partially resisted mild methylation. Sulfated MS were limited to primitive endocardial cells which gave origin to cushion tissue. Dye deposits of aldehyde fuchsin, high iron diamine or Alcian Blue (pH 1.0) were localized on cell surfaces and such staining was prevented by strong (60°C) methylation. Hyaluronidase sensitivity of sulfated MS decreased with gestation. The critical electrolyte concentration varied from 0.5-0.7 M MgCl2 on days 11½-12½ to 0.8-0.9 M MgCl2 after day 12½. The sulfated MS of endocardial cells were preceded by a transitory accumulation of diastase-resistant, periodic acid-Schiff-positive material. Possible roles of MS in normal and abnormal cardiac septation processes are discussed.

Effect of pepsin on partially purified pig gastric mucus and purified mucin

1988 ◽  
Vol 66 (5) ◽  
pp. 367-373 ◽  
Author(s):  
Sum P. Lee ◽  
Jane F. Nicholls ◽  
Anthony M. Roberton ◽  
Han Z. Park
Keyword(s):  
Periodic Acid ◽  
Gastric Mucin ◽  
Low Density ◽  
Gastric Mucus ◽  
Experimental Conditions ◽  
Periodic Acid Schiff ◽  
Pepsin Digestion ◽  
Cscl Density Gradient ◽  
Peptic Digestion

Partially purified native-pig gastric mucus and purified pig gastric mucin, prepared by column chromatography and caesium chloride (CsCl) density-gradient ultracentrifugation, were subjected to pepsin digestion. The products of peptic digestion were chromatographed on Sepharose CL-2B, and fractions were assayed for carbohydrate by the periodic acid – Schiff reaction. The polymeric gastric mucin in the purified mucin samples was readily degraded by pepsin. In sharp contrast, the polymeric mucin in the partially purified mucus was relatively resistant to pepsin digestion. In 45 min, pepsin degraded 40% of the polymeric mucin in the purified samples, whereas it produced no significant degradation (<10%) in the partially purified mucus samples. In partially purified gastric mucus, treated with CsCl but not fractionated by ultracentrifugation, digestion with pepsin was also slow and incomplete. This showed that differences in susceptibility between partially purified and purified preparations are not due to the chaotropic effects of CsCl. In addition, the recombination of low-density nonmucin fractions in CsCl ultracentrifugation with the mucin also resisted pepsin digestion. Finally, we have shown that the low-density fractions in mucus exhibited a strong inhibitory effect of peptic activity in vitro. We conclude that under our experimental conditions, pepsin has little effect on partially purified mucus, and our findings indicate an inhibitor of peptic digestion is present in native gastric mucus. It is likely, but unproven, that this inhibitor is a noncovalently bound lipid present in the low-density fraction.

Clarithromycin inhibits overproduction of muc5ac core protein in murine model of diffuse panbronchiolitis

2003 ◽  
Vol 285 (4) ◽  
pp. L847-L853 ◽  
Author(s):  
Yukihiro Kaneko ◽  
Katsunori Yanagihara ◽  
Masafumi Seki ◽  
Misuzu Kuroki ◽  
Yoshitsugu Miyazaki ◽  
...  
Keyword(s):  
Core Protein ◽  
Periodic Acid ◽  
Protein A ◽  
Mitogen Activated Protein Kinase ◽  
Periodic Acid Schiff ◽  
Western Blots ◽  
Diffuse Panbronchiolitis ◽  
Mucin Production ◽  
Long Term Treatment

Long-term treatment of macrolide antibiotics is considered an effective treatment for diffuse panbronchiolitis (DPB). Although hypersecretion is a common feature of this disease, and it is known that macrolides inhibit mucin production, the mechanism of the effect on mucin production is unclear. The aim of our study was to determine the production of muc5ac core protein, a major core protein of mucin in airway secretion, and the effect of clarithromycin treatment on such production in a mouse model mimicking DPB. Alcian blue-periodic acid-Schiff-positive cells were detected in the lungs of Pseudomonas aeruginosa-infected mice. Western blots of these mice showed muc5ac glycoprotein at day 1 and increased progressively from day 4 to day 14 after inoculation of bacteria. Clarithromycin (10 mg · kg-1· day-1for 7 days) significantly reduced the muc5ac expression at both the mRNA and protein levels. To investigate the role of molecules upstream in muc5ac regulation, we examined the role of mitogen-activated protein kinase. Extracellular signal-regulated kinase 1/2 phosphorylation increased in the infected lung and decreased after treatment. Our results suggest that overproduction of muc5ac plays an important role in the pathogenesis of DPB and that clinical improvement following macrolide therapy seems to involve, at least in part, its inhibition of mucin overproduction, through modulation of intracellular signal transduction.

scholarly journals Purification and properties of plaice metallothionein, a cadmium-binding protein from the liver of the plaice (Pleuronectes platessa)

1979 ◽  
Vol 183 (2) ◽  
pp. 277-283 ◽  
Author(s):  
J Overnell ◽  
T L Coombs
Keyword(s):  
Positive Reaction ◽  
Binding Protein ◽  
Periodic Acid ◽  
Molar Ratio ◽  
Amino Acid Residues ◽  
Thiol Groups ◽  
Single Chain ◽  
Periodic Acid Schiff ◽  
Pleuronectes Platessa ◽  
Mammalian Metallothioneins

A low-molecular-weight protein induced in the liver of the plaice (Pleuronectes platessa) by exposure to cadmium was purified and characterized. It is closely similar to mammalian metallothioneins in all of its properties in that it is a single-chain cadmium-binding protein of approx. 7000 mol.wt. with a high cysteine content (31 mol%) and no aromatic amino acid residues. The thiol groups of the cysteine residues complex with the cadmium in a SH/Cd molar ratio of 3:1 and produce a characteristic absorption maximum at 250 nm. Unlike the mammalian metallothioneins, however, metal analyses reveal only traces of zinc and copper in addition to cadmium. The presence of carbohydrate previously assumed from a positive reaction with periodic acid/Schiff reagent has now been disproved, and the positive reaction attributed to interaction with the thiol groups in the protein.

scholarly journals The degradation of cartilage proteoglycans by tissue proteinases. Proteoglycan structure and its susceptibility to proteolysis

1977 ◽  
Vol 167 (3) ◽  
pp. 629-637 ◽  
Author(s):  
P J Roughley ◽  
A J Barrett
Keyword(s):  
Ionic Strength ◽  
Cathepsin D ◽  
Core Protein ◽  
Degradation Products ◽  
Gradient Centrifugation ◽  
Limited Proteolysis ◽  
Extraction Procedure ◽  
Cathepsin G ◽  
Nasal Cartilage ◽  
Cscl Density Gradient

1. Proteoglycan was obtained from bovine nasal cartilage by a procedure involving sequential extraction with a low-ionic-strength KCl solution, then a high-ionic-strength CaCl2 solution. Purification was by CsCl-density-gradient centrifugation. 2. The CaCl2- extracted proteoglycan was subjected to proteolytic degradation by papain, trypsin, cathepsin D, cathepsin B, lysosomal elastase or cathepsin G. Degradation was allowed to proceed until no further decrease in viscosity was detectable. 3. The size and chemical composition of the final degradation products varied with the different proteinases. Cathepsin D and cathepsin G produced glycosaminoglycan-peptides of largest average size, and papain produced the smallest product. 4. The KCl-extracted proteoglycan was intermediate in molecular size and composition between the CaCl2-extracted proteoglycan and the largest final degradation products, and may have been formed by limited proteolysis during the extraction procedure. 5. It is postulated that the glycosaminoglycan chains are arranged in groups along the proteoglycan core protein. Proteolytic cleavage between the groups may be common to the majority of proteinases, whereas clevage within the groups is dependent on the specificity of each individual proteinase.

Isolation, culture and characterization of chicken primordial germ cells

2006 ◽  
Vol 3 (3) ◽  
pp. 183-188 ◽  
Author(s):  
Tang Xin-Yan ◽  
Zeng Wei-Dong ◽  
Mi Yu-Ling ◽  
Liu Hong-Yun ◽  
Zhang Cai-Qiao
Keyword(s):  
Germ Cells ◽  
Gradient Centrifugation ◽  
Periodic Acid ◽  
Primordial Germ Cells ◽  
Culture Conditions ◽  
Nuclear Antigen ◽  
Periodic Acid Schiff ◽  
Ficoll Density Gradient Centrifugation ◽  
Pas Staining ◽  
Proliferating Activity

AbstractPrimordial germ cells (PGCs) were isolated from the genital ridges of chicken (Gallus domesticus) embryos at the 19th stage and purified by Ficoll density-gradient centrifugation. PGCs were co-cultured with somatic cells in preliminary culture and subcultured. Identification of PGCs was carried out by histochemical methods, including alkaline phosphatase (AKP) and periodic acid–Schiff (PAS). The proliferating activity of PGCs in subculture was demonstrated by immunocytochemistry of proliferating cell nuclear antigen (PCNA). Meanwhile, proliferating PGCs were compared under different culture conditions of 5–20% fetal cattle serum (FCS), insulin–transferrin–selenite (ITS) medium, conditioned medium (CM), 15% FCS+ITS, 15% FCS+40% CM. The results showed that the cultured PGCs were positive for AKP and PAS staining and displayed intensive proliferating activity by PCNA. The PGCs without centrifugation grew better than those with centrifugation. The PGCs formed larger colonies in media with 5% FCS or ITS than other media, indicating that 5% FCS or ITS supplemented media could be an ideal culture system for PGC proliferation in the PGC-somatic cell co-culture, in addition to the embryonic fibroblast feeder layer.

Isolation and partial characterization of a soluble mucin in human pancreatic juice

1991 ◽  
Vol 69 (8) ◽  
pp. 566-571
Author(s):  
S. P. Lee ◽  
Y. S. Choong ◽  
H. Z. Park
Keyword(s):  
Molecular Weight ◽  
Pancreatic Juice ◽  
Periodic Acid ◽  
Average Molecular Weight ◽  
Small Molecular Weight ◽  
Periodic Acid Schiff ◽  
Cellulose Acetate Electrophoresis ◽  
Cscl Density Gradient ◽  
Oligosaccharide Moiety ◽  
Schiff Reagent

Morphological and histochemical abnormalities in pancreatic mucin occur in many pancreatic disorders. However, the composition of pancreatic mucin is poorly understood. Purified mucin was isolated from pure pancreatic juice by sequential chromatography on Sepharose CL-2B and CL-4B followed by CsCl density gradient ultracentrifugation. The mucin preparation consists of 24% protein and 73% carbohydrate. Reduction of the macromolecule (> 2 × 106) by mercaptoethanol resulted in the formation of subunits of molecular weight 500 000 and released several small molecular weight proteins, including a glycoprotein of an average molecular weight of 116 000. Cellulose acetate electrophoresis separated the mucin into three species of different staining properties for periodic acid-Schiff reagent and Alcian blue, suggesting the presence of microheterogeneity with respect to sulphation and sialation. Threonine, serine, and proline composed 48% of the total amino acids, while the oligosaccharide moiety contained N-acetylglucosamine, N-acetyl-galactosamine, fucose, galactose, sialic acid, and sulphate. We also detected the presence of C16:0 and C18:0 fatty acids which were probably noncovalently bound to the pancreatic mucin.Key words: pancreas, mucin, glycoprotein, cystic fibrosis.

scholarly journals The presence of a cartilage-like proteoglycan in the adult human meniscus

1981 ◽  
Vol 197 (1) ◽  
pp. 77-83 ◽  
Author(s):  
P J Roughley ◽  
D McNicol ◽  
V Santer ◽  
J Buckwalter
Keyword(s):  
Hyaluronic Acid ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Large Subunit ◽  
Dermatan Sulphate ◽  
Keratan Sulphate ◽  
Adult Human ◽  
Cscl Density Gradient ◽  
Cartilage Proteoglycans ◽  
Aggregate Structures

Proteoglycans were extracted from the adult human meniscus under dissociative conditions and purified by CsCl-density-gradient centrifugation. The preparations of highest density contained proteoglycan that possessed the ability to interact with hyaluronic acid, was of large subunit size and was composed of chondroitin sulphate, keratan sulphate and sialic acid-containing oligosaccharides. This ‘cartilage-like’ proteoglycan also exhibited subunit and aggregate structures analogous to those of hyaline-cartilage proteoglycans when examined by electron microscopy. However, the composition of this proteoglycan was more comparable with proteoglycans from immature cartilage than from age-matched cartilage. The preparations from lower density, which were enriched in dermatan sulphate, contained smaller proteoglycan that was not able to interact with hyaluronic acid. This non-aggregating proteoglycan may be structurally distinct from the ‘cartilage-like’ proteoglycan, which does not contain dermatan sulphate.

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